4.5 (in-situ); 2.3 (on-deck)

in-situ levels; 75, 54, 36, 10, 1 and 0.1% surface irradiance level

5 mL of 15N2 gas (99 atom% 15N) (in-situ); 2.5 mL of 15N2 gas (on-deck)

12 mL of each 4.5 bottle subsampled in Exetainers

fixed with HgCl2 and stored upside down at 4oC in the dark, analyzed within 6 months after the cruise

25 mm, 0.7 um porosity, Whatman GF/F filters, pre-combusted (450oC, 4 h), stored frozen at -20oC, then dried at 60oC for 24h before analysis

EA-IRMS Integra2 Sercon Ltd

(used 14C tracer method of Moutin and Raimbault, 2002)

HOT program time-series cruise to Station ALOHA

15N incubation (bubble method and 15N enriched water)

Montoya et al., 1996 and Wilson et al., 2012

acid-washed polycarbonate bottles/caps fitted with silicone septa

in-situ levels and in the shade

3 mL of 15N2 (bubble method); 100 mL of 15N2-enriched water

Cambridge Isotope Laboratories between June 2005 and September 2009; Sigma-Aldrich between November 2009 and May 2012; Sigma-Aldrich between June 2012 and September 2013; and Cambridge Isotope Laboratories between October and December 2013.

Sigma-Aldrich lots SZ1670V, EB1169, and CX0937 and Cambridge Isotope Laboratories Lot I-16727

2.8 and 3 atom% at beginning of incubations with enriched water

25 mm GF/F filters, stored frozen at -20oC

Carlo-Erba EA NC2500 coupled with ThermoFinnigan Delta S

1) Eastern South Pacific, BIG RAPA (R/V Melville), 2) Pacific North-west California Current system (Fairweather and R/V Point Sur), 3) Station ALOHA (R/V Kilo Moana)

November to December 2010 (Eastern South Pacific); August 2012 (Pacific North-West California Current system; March 2014 (Station ALOHA)

15N incubation (15N enriched water)

Mohr et al., 2010; Wilson et al., 2012; Montoya et al., 1996

acid-washed and milli-Q rinsed polycarbonate bottles (1.1 or 2.2 L for PNW incubations, and 4.4 L for ESP incubations)

either in the dark of with screening to mimic approximate in situ light conditions

350 mL (ESP incubations) or 200 mL (PNW incubations) of 15N2-enriched seawater (5 mL 99 atom% in 500 mL)

99.0 atom% Cambridge Isotopes

25 mm GF/F filter, pre-combusted, frozen at -80oC

0.00146 atom % (Montoya et al., 1996)

0.15 to 0.77 nmol N L-1 d-1 (ESP), 0.04 to 1.08 nmol N L-1 d-1 (NPSG), 0.06 to 0.69 nmol N L-1 d-1 (PNW)

0.369 to 0.389 (T = final, average)

26 ± 0.1oC, and 27oC

Stylophora pistillata samples

15N incubation (15N enriched water addition)

Mohr et al., 2010; Grobkopf et al., 2012

glass containers, polycarbonate

20% volume with 15N-enriched seawater

pre-combusted (450oC, 5 h) GF/F filter

EA-IRMS, Integra CN, SerCon Ltd, Cheshire, UK

VAHINE (Variability of vertical and trophic transfer of diazotroph derived N in the souh west Pacific), oligotrophic New Caledonian lagoon

15N incubation (15N enriched water)

Mohr et al., 2010; Wilson et al., 2012

in-situ (mesocosm experiments)

polycarbonate bottles

5% vol/vol of 15N2-enriched seawater (1 mL of 15N2 98.9 atom% per 100 mL of seawater)

98.9 atom%, Cambridge Isotope Laboratories

2.4 ± 0.2 at % (n = 10)

12 mL subsampled in exetainers

preserved upside down in the dark at 4oC and analyzed less than 6 months after the experiment

25 mm GF/F filter, pre-combusted (4 h at 450oC), 0.7 um nominal porosity, frozen at -20oC

Delta Pkus Thermo Fisher Scientific IRMS coupled with a Flash EA, Thermo Fisher Scientific

15N enrichment of the PON higher than 3 times the standard deviation obtained from T0 samples

26/27 June, 17/18 July, 06/07 Aug 2012; 18/19 June, 17/18 July, 14/15 Aug 2013

15N incubation (15N enriched in-situ water)

Montoya et al., 1996; Mohr et al., 2010

2012: 50 mL of 15N-enriched water (1 mL 15N2 per 100 mL water); 2013: 50 mL 15M-enriched water (2.4 mL 15N2 per 100 mL)

yes, gently inverted 20-times by hand

1-1.5% in 2012 and 10-11% in 2013

headspace-free 12 mL exetainer vials

preserved with 100 uL saturated ZnCl2 solution

25 mm GF/F Whatman filter, pre-combusted, frozen at -80oC, and freeze-dried

January to February 2010; March to April 2011

15N incubation (bubble method)

acid-washed, sample-rinsed, light transparent polycarbonate

simulated in-situ levels

1.5 mL of 99 atom% 15N2

SZ1670V and MBBB0968V

25 mm Whatman glass fiber filter, precombusted

VAHINE (Variability of vertical and trophic transfer of diazotroph derived N in the souh west Pacific), oligotrophic New Caledonian lagoon

13 January 2013 to 4 February 2013

15N incubation (15N enriched in-situ water)

in-situ (mesocosm experiments)

polycarbonate bottles

1:20 (vol:vol) of 15N2-enriched seawater (1 mL of 98.9% per 100 mL)

98.9% Cambridge Isotopes

2.4 ± 0.2 atom% (n=10)

12 mL of incubated water sampled on Exetainers

GF/F filter, combusted, stored at -20oC, dried at 60oC for 24 hr prior to analysis

Delta Plus coupled to a Flash EA (Thermo Fisher Scientific)

transect crossing North Atlantic to the Bahamas onboard R/V Sarmiento de Gamboa

15N incubation (bubble method and 15N enriched water)

Montoya et al., 1996 and Mohr et al., 2010

transparent polycarbonate (Nalgene)

covered with neutral density screens (Lee Filters)

200 mL of 15N2-enriched seawater (5 mL 99 atom% in 500 mL)

99 at % 15N, Cambridge Isotope Laboratories

25 mm GF/F filters, precombusted, stored in cryovials and frozen.

Gulf of California and Eastern Tropical North Pacific

summers of 2004, 2005 and 2008

between surface and 40-60

free-floating array, Niskin on CTD rosette

15N incubation (bubble method)

see White et al. (2007)

see White et al. (2007)

see White et al. (2007)

see White et al. (2007)

see White et al. (2007)

see White et al. (2007)

25 mm diameter, precombusted (450oC for 12 h) glass fiber filters (GF/F), stored at -20oC, dried overnight at 60oC, acidified before analysis

see Prahl et al. (2005)

North American coastal waters between Cape Hatteras and Georges Bank/R/V Hugh Sharp (summe and automn 2006) and R/V Delaware II (August 2009)

summer and fall 2006 and August 2009

36.5°N and 39°N; 35°N and 43°N

(-76°W and -74°W; -76°W and -65°W)

upper 6 m, between and near the bottom

15N incubation (bubble method)

Montoya et al., 1996; Mulholland et al., 2006

incubation bottles and clean carboys

<10% of 99% 15N gas

GF/F filter, pre-combusted (450oC for 2 h), stored frozen and dried before analysis

October 2010 and December 2011

Water-column samples, Oligotrophic North Pacific Ocean

C2H2 and 15N incubation (15N enriched water addition and bubble addition)

Montoya et al., 1996 and Mohr et al., 2010

polycarbonate bottle

in-situ levels and 50, 25 and 10% of full sunlight

50 mL of 15N-enriched seawater from 70 ml crimp sealead vials with enriched seawater from surface waters at station ALOHA prepared on land added to , no headspace. Samples for bubble method were injected with 3 mL of 15N2 gas (98 atom%).

98 atom%, Sigma-Aldrich)

Y. Inverted 20 times (enriched 15N water) and gently shaken (bubble method)

1.5 atom% at beginning of incubations with enriched water.

15N2 was NOT measured after incubation. Replicate samples of prepared 15N2-enriched seawater were analyzed for the loss of 15N2 at weekly intervals over a 1-month period.

Compare the ratio of mass 30 in 15N2 enriched seawater/reference seawater at 25oC.

25 mm glass fiter filters combusted (450oC for 5h), stored frozen at -20oC

EA-IRMS Carlo Erba NC2500 coupled to Thermo-Finnigan Delta S

Wilson et al., Applied and Environmental Microbiology, 2012

Subtropical northeast Atlantic/project 'Shelf-Ocean Exchanges in the Canaries - Iberian Large Marine Ecosystem' (CAIBEX), R/V 'Sarmiento de Gamboa

15N incubation (bubble method) and C2H2 reduction experiments

Stal, 1988 and Montoya et al., 1996

Polycarbonate (Nalgene)

incident PAR light at 5 m depth (Neutral density screens - Lee Filters)

precombusted GF/F filters, wrapped in aluminium foil, and storel at -20oC until analysis

Thermo Flash EA 1112 elemental analyser interfaced by a Conflo III with a Thermo Delta V Advantage IRMS

0.001 nmol N l-1 d-1 (considering a minimum acceptable change of d15N between the initial and the final PON sample of 4 ‰, the incubation time and the detection limit of the elemental analyser used (0.75 ug N))

Mediterranean Sea/BOUM cruise onboard the R/V Atalante

15N incubation (bubble method)

acid-washed polycarbonate

75%, 50%, 35%, 20%, 10%, 3% 1%, 0.3% and 0.1% surface irradiance levels

4 mL of 99% 15N2 gas

25 mm, 0.7 um nominal porosity GF/F pre-combusted (4 h at 450oC) glass fiber filters, dried at 60oC, then stored over dessicant until analysis

0.08 to 0.15 umoles (3 times the standard deviation on 10 blanks analysis)

0.10 to 0.19 umoles N (10 times the standard deviation on 10 blanks analysis)

0.45 to 3.65 umoles N

Eastern Arabian Sea/cruise #SK 258 on board ORV Sagar Kanya

CTD rosette fitted with Go-Flo bottles/care was taken to avoid trace metal contamination

15N incubation (bubble method)

polycarbonate (Nalgene, USA)

99% Cambridge Isotope Laboratories, Massachusetts, USA

25 mm, 0.7 um nominal porosity GF/F pre-combusted (4h at 400 oC) Whatmann glass fiber filters and dried in an oven at 50oC overnight and stored until analysis

Flash EA 1112 series, CE instruments, Italy, interfaced with a Finnigan Delta Plus Continuous Flow Mass Spectrometer

two hypoxic basins in the Southern California Bight/San Pedro Ocean Time Series (SPOTS) station (RV Sea Watch), Santa Monica Bay Observatory (SMBO) station (RV Seaworld)

July 2004 to September 2006 (SPOTS), July 2006 to July 2007 (SMBO)

surface, chlorophyll maximum (range 16 to 37 m), 500 m, and 885 m (ca. 1.5 m from bottom) (SPOTS),

15N incubation (bubble method)

4 (SPOTS), 2.5 (SMBO)

ambient light, 1% of surface, and dark

trace additions of 98 atom% 15N2

98 atom%, Sigma-Aldrich

Nucleopore prefilters (Whatman) of poresize 10 um followed by precombusted GF/F (poresize 0.7 um, Whatman) or on some dates, through GF/F filters alone, dried for 24 h at 60oC

SerCon Integra, University of California Davis, Stable Isotope Facility

Atlantic Ocean/TRYNITROP (Trichodesmium and N2 fixation in the Atlantic Ocean) cruise

surface, deep chlorophyll maximum, and an intermediate depth between both

15N incubation (bubble method)

25 mm GF/F filter (Whatman), dried at 40oC for 24 h, and stored until analysis

Flash EA112 + Deltaplus Thermo Finnigan

acetanilide standard

(14C method used, separate incubations)

Mourino-Carballido et al, Limnology and Oceanography, 2011

North Pacific subtropical and subarctic gyres (Pacific Open Ocean Bloom cruise) and near the Hawaiian islands (Ocean PERturbation Experiment cruise, OPEREX)

28°N to 32°N 22°N to 26°N

15N incubation (bubble method)

Acid-washed polycarbonate bottles

10 and 60% irradiance

99 atom% Cambridge Scientific

Y. Inverted several times

25 mm pre-combusted glass fiber filter, stored at -20oC, dried overnight at 60oC before analysis

PDZ Europa ANCA-GSL elemental analyzer interfaced to a PDZ Europa 20-20 IRMS

Watkins-Brandt et al., Marine Ecology Progress Series, 2011

Atlantic Ocean/TRYNITROP (Trichodesmium and N2 fixation in the Atlantic Ocean) cruise, on board BIO Hesperides

November-December 2007 and April-May 2008

26°S to 33°S (2007) and 29°S to 31°S (2008)

surface (5), an intermediate depth (30-80), and the depth of the deep chlorophill maximum

15N incubation (bubble method)

Montoya et al., 1996 with modifications described in Rees et al. (2009)

acid-cleaned clear polycarbonate (Nalgene)

in-situ levels (simulated by blue (Mist Blue, Lee filters) and neutral density screens)

Whatman GF/F filter (25 mm in diameter), dried at 40oC during 24 h and stored at room temperature until analysis

FlashEA112 + Deltaplus Thermo Finnigan

(acetanilide standard)

North Pacific subtropical gyre/Station ALOHA, instrumented physical and biogeochemical bottom-moored platforms, HALE ALOHA (Hawaii Air-sea Logging Experiment A Long-term Oligotrophic Habitat Assessment) and WHOTS (Woods Hole Hawaii Ocean Time-series Station)

August 2004 to July 2007 (HALE ALOHA), August 2004-2007 (WHOTS)

15N incubation (bubble method)

98% Isotech Laboratories, Inc.

precombusted 25 mm glass fiber filters, dried for 24 h

EA-IRMA Carlo Erba NC2500 coupled to a Thermo-Finnigan Delta S)

Tropical and subtropical western North Pacific/MR04-07 and MR05-02 cruises on board the R/V Mirai

November to December 2004 and May to June 2005

HCl-rinsed bucket and Niskin-X samplers mounted on CTD-carousel

C2H2 reduction experiments

Capone and Montoya, 2001)

HCl-rinsed polyethylene-terephthalate

50%, 25%, 10% and 1%

Oligotrophic North Pacific Ocean/close proximity to stn ALOHA

15N incubation (bubble method)

30% incident irradiance

3 mL of 98% 15N gas

25 mm GF/F pre-combusted glass fiber filters and dried

EA-IRMS Carlo Erba NC2500 coupled to a Thermo-Finnigan Delta S

Stn ALOHA/HOT program (cruises 158 to 159, 161 to 165 and 167 to 168)

April to May 2004, July to November 2004, and February to March 2005

stringent trace metal clean procedures were not employed

15N incubation (bubble method)

Montoya et al., 1996; Capone & Montoya, 2001

acid-washed Tedlar gas sampling bags fitted with gas-tight Teflon-backed septa and polycarbonate bottles sealed with caps fitted with thick silicone septa (cruises 167 and 168)

4.7 (bags), 4.5 (bottles)

simulated in-situ levels

GFF filter and 10 um Nitex mesh filtrate also filtered onto a GFF filter, stored frozen (-20oC), until analysis

Carlo-Erba Elemental Analyzer NC2500 interfaced with a Finnigan delta S ion ratio mass spectrometer

0.06 umol N m-3 d-1 (analytical capability of the instrument - calculated for the smallest PN masses sampled, using twice the conservative upper estimate of instrument precision, 0.8 ‰)

0.03 umol N m-3 d-1 (calculated from twice the average standard error of triplicate T0 values (twice 0.32‰) and the average PN mass sampled)

New Caledonia (Southwest Pacific) and Noumea lagoon/ 6 Diapalis cruises on the 'Institut pour la Recherche et le Developpement' (IRD) ship 'l"Alis'

January 2002 and October 2003; October 2005

0, 10, 20, 30, 40, 80, 100; between 0 and 20 m in the lagoon

15N incubation (bubble method)

Polycarbonate Nalgene Flasks

2 mL of 99 atomic% 15N2 gas

pre-combusted (24 h at 450oC), 25 mm Whatman GF/F filter, dried in a 60oC oven for 24 h and analysed as soon as possible after the cruise

Integra-CN PDZ Europa mass spectrometer

>0.014% enrichment (2 times the standard deviation obtained with 8 time=0 samples) or 0.3 nmol l-1 12 h-1

0.3663 ± 0.007% (8 samples filtered immediately after isotope addition)

0.2 to 10 umol N, 0.3672 to 0.3681 at % (glycine); 5.23 umol N, 47.3 ± 0.27 at % (Urea, IAEA 310A); 4.49 umol N, 243 ± 0.46 at % (Urea, IAEA 310B)

Niskin on CTD rosette (trace-metal clean)

15N incubation (bubble method)

trace-metal clean polycarbonate (Nalgene)

40% of surface irradiance

Cambridge Isotope Laboratories

25 mm GF/F Whatman filters, precombusted at 450oC for 4.5 h, dried at 60oC and stored in a dessicator

Y (sealed in a dessicator containing an open beaker of 100% HCl for 36 h)

Europa Integra continuous flow mass spectrometer (University of California Davis, Stable isotope Facility)

0.01315-0.03179 15N atom % (range in the difference between the time-zero samples and the incubation experiment samples)

0.36773 ± 0.0016 15N atom % (mean and standard deviation of glycine standards)

Tropical North Atlantic (May-June 1994 on R.V. Gyre, March-April and October-November 1996, January-February 2001, and April-May 2003 on R.V. Seward Johnson, and July-August 2001 on R.V. Knorr)

May-June 1994, March-April and October-November 1996, January-February 2001, April-May 2003, and July-August 2001.

Trichodesmium colonies diluted in surface seawater

net tows and Niskin on CTD rosette

C2H2 reduction experiments and 15N incubation (bubble method)

Capone, 1993; Montoya et al., 1996

acid-washed serum vials (C2H2 method), glass bottle with screw cap seals with rubber septa (15N-N2 bubble method)

14 mL (C2H2 method), 310 mL (15N-N2 bubble method)

100%, 55%, 28%, 10% and 1% of surface irradiance

99 atom% Cambridge Isotopes

45 mm GF/F filter, pre-combusted (2 h at 450oC), dried at 60oC and stored over dessicant

Carlo Erba elemental analyzer interfaced to a Micromass Optima mass spectrometer

Tropical North Atlantic and Tropical North Pacific Oceans/

summer 2001 and spring 2002, fall 2002

7° 10'23"N to 12° 13'78"N; 22° 75'N

45° 29'60"W to 55° 55'38"W; 158° 00'W

surface, 25, 50, 100, and 150

Cell concentrates diluted 0.2 um pore size filtered seawater (final enrichment = 130 to 160 fold)

15N incubation (bubble method) and C2H2 reduction experiments

Capone, 1993 and Capone and Montoya, 2001

100, 50, 25, and 1%

combusted glass fiber filter, folded and left to dry in oven at 60oC before analysis

Falcon et al., Applied and Environmental Microbiology, 2004

Oligotrophic Pacific Ocean/ Hawaii Ocean Timeseries station ALOHA (cruise EW-9912) and along a transect from Hawaii to San Diego (cruise Cook-25)

surface, 25, mixed layer and pigment maximum

Water-column samples filtered through 100 um Nitex mesh to exclude large organisms, including Trichodesmium colonies and large diatoms such as Hemiaulus that might contain endosymbiotic N2 fixers

15N incubation (bubble method)

polycarbonate equipped with silicone septum caps

trace additions of 99 atom% 15N2

99 atom%, Cambridge Isotope

precombusted GF/F filter after passage through a 10-um prefilter to remove larger phytoplankton, dried at 60oC, then stored over dessicant until analysis

Micromass Optimas mass spectrometer interfaced to a CE Elantech NA2500 elemental analyser

Trichodesmium colonies from water-column samples

surface drift tows, Niskin and Go-Flo

15N incubation (bubble method) and C2H2 reduction experiments

14 mL serum vial filled with GF/F filtered seawater

in situ levels at 10 m depth; 9 to 60% of ambiant surface light intensity

0.2 mL of 98% of 99% 15N2

98% Cambridge Isotope Laboratories, Cambridge, MA; generated from 15N-labelled (99%) ammonium by treatment with alkaline hypobromite (Bremner, 1965)

7 mm precombusted Whatman GF/C filters, stored at -20oC, dried at 60oC before analysis

Europa 20-20 stable isotope analyzer equipped with ANCA-SL preparation unit, Crewe, UK

(measured in separate incubations)